Process for producing asialo GM1

ABSTRACT

The present invention provides a process for preparing asialo G M1  characterized in that neuraminidase isozyme L is allowed to act on gangliosides to produce asialo G M1 .

Technical Field

This invention relates to a process for producing asialo G_(M1).

2. Prior Art

"Ganglioside" is a general name of the group of sialic acid-containing sphingoglycolipids and is known to exist in the brain of higher animals, especially in the nervous system. In the living body, the gangliosides not only take part in nervous function and recognition, differentiation, proliferation, canceration, senescence, etc. of cells, but also, from a cell sociological view point, are responsible for receptor function of cytotoxins such as cholera toxin and botulinum toxin, peptide hormones such as thyroid hormone, interferons and so on. The gangliosides are also supposed to contribute to negative charges on the surface of cells. In fact, it is reported that the gangliosides extracted from bovine brain promote regeneration of damaged peripheral nerve and useful for treatment of alcoholic polyneuritis, diabetic neuropathy, etc (Iyaku to Yakugaku, 13(6), pp1407-1412 (1985); Practice, 4(4), pp 452-456 (1987); Iyaku Journal 22(7), pp 55-62 (1986)).

As described above, gangliosides are important substances having various functions in the living body. Therefore, it is desired to establish a process for collecting each of various gangliosides in a pure form. In order to prepare a specific ganglioside, conventionally attempted are methods using various kinds of chromatography. However, since the starting material, i.e., ganglioside components as extracted from bovine brain or the like is a ganglioside mixture containing various gangliosides each in trace amounts, it is difficult to obtain the desired products even by conducting cumbersome procedures, and any proper purification methods have not been found yet. Further, although an attempt is made to synthesize gangliosides by chemical technique, its process is complicated and side reactions are likely to occur, entailing difficulty in removing by-products.

On the other hand, neuraminidase is an enzyme which has been considered not to act directly on the gangliosides on its own. As a matter of fact, it is only reported that asialoganglioside G_(Al) is formed from ganglioside G_(M1) in the presence of detergents such as cholates (Masaki Saito, Taisha, 16, p761 (1977)). Moreover, it has been unknown at all that any isozyme of the neuraminidase exists.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a process for collecting a specific ganglioside, i.e., asialo G_(M1) from gangliosides.

Another object of the present invention is to provide a process for producing asialo G_(M1) in high purity and in good yield.

Namely, the present invention provides a process for producing asialo G_(M1) characterized in that neuraminidase isozyme L is allowed to act on gangliosides to obtain asialo G_(M1).

The present inventors have conducted intensive research and consequently found that the neuraminidase derived from the bacteria of the genus Arthrobacter has a novel isozyme and that, when said neuraminidase isozyme L is allowed to act on gangliosides, asialo G_(M1) can be selectively obtained with high purity and in good yield. According to the process of the present invention, by-products are not produced, and therefore asialo G_(M1) obtained can be readily purified. Further, the present invention has the advantage that asialo G_(M1) can be prepared using said enzyme in the absence of any detergent which was used in the conventional process.

The neuraminidase isozyme L (hereinafter referred to as "Isozyme L") to be used in the present invention has the following physicochemical properties :

(1) Action: Selectively producing asialo G_(M1) from gangliosides.

(2) Molecular weight: About 88000 dalton

The molecular weight was determined according to gel filtration chromatography and SDS-PAGE electrophoresis as described below.

Gel filtration chromatograph

Gel filtration method was done using Sephadex G150 (product of Pharmacia). As the eluent, 50 mM phosphate buffer (pH 6.8) was used.

SDS-PAGE electrophoresis

SDS-polyacrylamide gel electrophoresis was conducted according to the method of U. K. Laemmli (Nature, 227, 680 (1970)).

(3) Optimum pH: 4.7-5.5 (when bovine brain gangliosides used as the substrate)

(4) Stable pH range : 4.5-9.5

(5) Optimum temperature: 45-55° C.

(6) Thermal stability: not higher than 60° C.

On the other hand, the neuraminidase derived from Arthrobacter ureafaciens was divided into type I and type II. Their physicochemical properties are as described in TABLE I below (Y. Uchida et al., J. Biochem., 86, 425, (1979)).

                  TABLE I                                                          ______________________________________                                                         I        II                                                    ______________________________________                                         Molecular Weight  51000      39000                                             (gel filtration method)                                                        Optimum pH        5.0-5.5    5.0-5.5                                           Stable pH range   6.0-9.0    6.0-9.0                                           Optimum Temperature                                                                              53° C.                                                                             53° C.                                     ThermaI Stability ≦50° C.                                                                     ≦50° C.                             ______________________________________                                    

The above description shows that Isozyme L is different from the conventional neuraminidase in physicochemical properties such as action, molecular weight, optimum pH, stable pH range, optimum temperature and thermal stability, and therefore they are enzymes of different molecular species.

The activity of Isozyme L is determined in the same manner as in the neuraminidase, for example, according to thiobarbituric acid method as described in J. Biol. Chem., 234, 1971 (1959)), etc. One unit of Isozyme L is defined as the amount of enzyme liberating 1 μmole of N-acetylneuraminic acid per minute at 37° C.

Isozyme L can be collected from the culture obtained by culturing a bacterium of the genus Arthrobacter.

As the bacteria of the genus Arthrobacter, known bacteria can be used. Examples thereof are strains of Arthrobacter ureafaciens such as Arthrobacter ureafaciens M1057 (FERM BP-1391).

The incubation of the bacteria of the genus Arthrobacter can be conducted using either conventional liquid or solid media, but it is generally advantageous to use a liquid medium. Incubation with shaking or incubation with aeration and stirring is preferably conducted in order to produce the desired enzyme. The media for the above incubation are not specifically limited, and there can be used various media containing nutrients and the like commonly used for incubating microorganisms. Examples of the nutrients are-sugars such as glucose, fructose, lactose, invert sugar, saccharified starch solution, colominic acid, sorbitol and glycerol; organic acids such as pyruvic acid, malic acid and succinic acid, nitrogen sources such as peptides, meat extract, yeast extract, casamino acid, urea, ammonium salts and nitrate salts; inorganic salts of phosphorus, magnesium, potassium, sodium, etc.; trace elements such as boron, copper, iodine, iron, manganese, zinc, cobalt, molybdenum; trace growth factors such as vitamins; and so on. Further usable as the above media are, for example, natural or semi-synthetic media containing extract or exudate of animal tissues. Examples of these media are those marketed under the name of "Todd Hewitt Broth medium", "Brain Heart Infusion medium", etc.

Incubation was conducted generally at a temperature of about 20 to about 40° C, preferably about 25 to about 30° C. and completed in about 10 to about 70 hours.

Isozyme L can be prepared from the culture solution of the bacteria of the genus Arthrobacter in a conventional manner. For example, the bacteria are separated and removed from the culture solution, and the obtained supernatant is subjected to purification procedure such as ammonium sulfate fractionation and affinity chromatography and, if desired, may be further subjected to purification steps such as ion-exchange chromatography, chromatofocusing and gel filtration.

By allowing the Isozyme L thus obtained to act on gangliosides, asialo G_(M1) is obtained selectively. Said enzyme reaction is conducted by adding Isozyme L to the starting solution containing gangliosides and buffer, and if desired, sterilized water.

In the above enzyme reaction, the gangliosides, i.e., the starting materials of asialo G_(M1) are not specifically limited and there can be used known gangliosides. Examples thereof are at least one member selected from gangliosides G_(M1), G_(Dla), G_(Dlb), G_(Tla), G_(Tlb) and G_(Qlb), or a mixture of gangliosides extracted from bovine brain, etc. The amount of gangliosides is not specifically limited, and generally it is not more than 50 mg, preferably about 0.05 to about 2 mg, per ml of the starting solution. As the buffer, conventional ones having a pH of about 4 to about 8 can be used and examples thereof are about 10 to about 200 mM acetate buffer (pH 4.5-5.5), McIlvaine buffer (pH 3.5-5.5), etc. The amount of Isozyme L is also not particularly limited and it is generally not less than 5 mU, preferably about 0.1 to about 10 U, per ml of the starting solution. The reaction temperature and pH are not limited so far as the enzyme is active, and generally the temperature can be about 20 to about 50° C. and pH can be about 4 to about 8. The reaction time is appropriately selected depending on the concentration of substrate, reaction temperature, etc. For example, the reaction time is 30 minutes to 24 hours at 37° C.

For purifying asialo G_(M1) from the reaction solution, known purification methods can be employed. For example, purification can be conducted by subjecting the reaction solution to solvent-extraction and then carrying out concentration, desalting, freeze-drying, etc.

EXAMPLES

The present invention will be described in greater detail with reference to the following reference example and examples.

REFERENCE EXAMPLE 1

In 2l-Sakaguchi flask was placed a medium (adjusted to pH 7.0) containing 5.0 g of lactose, 2.0 g of diammonium hydrogen phosphate, 3.0 g of sodium chloride, 1.0 g of dipotassium hydrogen phosphate, 0.1 g of magnesium sulfate and 1000 ml of deionized water. Then the medium was sterilized by heating in an autoclave.

This medium was inoculated with Arthrobacter ureafaciens M1057, followed by shaking culture at 28° C. for 24 hours. The culture solution obtained was centrifuged (10000 rpm, 10 minutes), giving a culture supernatant.

Subsequently, ammonium sulfate was added to the culture supernatant and the fraction precipitated when the ammonium sulfate was added to the supernatant to 80% saturation was collected. The precipitate was dissolved in a small amount of water, followed by dialysis against 10 mM acetate buffer (pH 4.5). The dialysate was passed through the column packed with the gel which was prepared by adding epichlorohydrin to 2N sodium hydroxide solution containing soluble starch and colominic acid and in which the colominic acid served as a ligand. The neuraminidase adsorbed was then eluted with 100 mM acetate buffer (pH 4.5).

The neuraminidase active fractions were subjected to salting-out with ammonium sulfate and dialysis against desalted water. The dialysate was separated into three components by chromatofocusing. The chromatofocusing was conducted as follows. The dialysate was passed through a column of polybuffer exchanger equilibrated with 25 mM imidazole-HCl (pH 7.4) using polybuffer 74 (product of Pharmacia) adjusted to pH 3.8 as eluent.

The three components obtained were independently subjected to salting-out with ammonium sulfate and dissolved in 100 mM phosphate buffer (pH 7.0). The solution was subjected to gel-filtration with Ultrogel AcA44 (product of I.B.F. Biotechnics), giving 4 components. Isozyme L (980 units) was obtained from one of these 4 components.

The enzyme activity of Isozyme L was determined according to thiobarbituric acid method as follows.

That is to say, 0.1 ml of the enzyme solution, 0.05 ml of 0.4% N-acetylneuraminyllactose solution and 0.05 ml of 0.2M acetate buffer (pH 5.0) were subjected to reaction at 37° C. for 10 minutes. The amount of N-acetylneuraminic acid released was determined by thiobarbituric acid method. One unit of Isozyme L was defined as the amount of enzyme producing 1 μmole of N-acetylneuraminic acid per minute at 37° C.

EXAMPLE 1

A 1 ml quantity of 0.5% ganglioside G_(Dla) solution, 1 ml of 40 mM acetate buffer (pH 5.0) and 1 ml of sterilized water were mixed together, and to the mixture was added 1 ml of aqueous solution of Isozyme L (8 U/ml). The mixture was allowed to react at 37° C. for 1 hour, and 1/10 part by volume of chloroform was added thereto to stop the enzyme reaction. Asialo G_(M1) was extracted from the reaction solution with use of a mixture of chloroform and methanol (2:1) and then lyophilized, giving 2.0 mg of asialo G_(M1) as a white powder.

A thin layer chromatographic analysis under the following conditions indicates that the white powder obtained above shows a single spot of the same Rf value (0.61) as the authentic sample of asialo G_(M1) (purity: not less than 99%, product of Honen). Therefore, it was confirmed that the product was asialo G_(M1) having a purity which is equal to or greater than that of the authentic sample.

Conditions of Thin Layer Chromatography

Thin layer plate: high performance TLC (HPTLC) No.5641 (product of Merck)

Developing solvent: mixture of chloroform, methanol and 0.02% CaCl₂ (60:35:8)

Color reagent: Orcin sulfuric acid reagent

EXAMPLE 2

A 10 ml quantity of 0.05% ganglioside G_(M1) solution, 10 ml of 40 mM acetate buffer (pH 5.0) and 10 ml of sterilized water were mixed together, and to the mixture was added 10 ml of aqueous Isozyme L (3 U/ml), followed by reaction at 37° C. for 8 hours. The reaction mixture was subjected to purification in the same manner as in Example 1, giving about 2.6 mg of asialo G_(M1) as a white powder. The production of asialo G_(M1) was confirmed in the same manner as in Example 1.

EXAMPLE 3

The reaction and purification were conducted in the same manner as in Example 2 except that 10 ml of a solution of ganglioside mixture (containing 5 mg of the gangliosides) obtained by extraction from bovine brain was used as the gangliosides, giving about 3.1 mg of asialo G_(M1) as a white powder. The production of asialo G_(M1) was confirmed in the same manner as in Example 1. 

We claim:
 1. A process for preparing asialo G_(M1) which comprises subjecting a solution of ganglioside or a mixture of gangliosides in the absence of detergent to the action of neuraminidase isozyme L obtained from a culture of bacterium of Arthrobacter ureafaciens M1057.
 2. A process according to claim 1 wherein the neuraminidase isozyme L has the following physicochemical properties:Action: Selectively producing asialo G_(M1) from gangliosides; Molecular weight about 88000 dalton (according to gel-filtration chromatography and SDS-PAGE electrophoresis); Optimum pH: 4.7-5.5 (when bovine brain gangliosides are used as the substrate); Thermal stability: 60° C. or lower.
 3. A process according to claim 1 wherein the ganglioside is at least one member selected from ganglioside on a mixture of gangliosides G_(Ml) G_(Dla), G_(Dlb), G_(Tla), G_(Tlb) and G_(Qlb) or a mixture of gangliosides extracted from bovine brain.
 4. A process according to claim 1 wherein the neuraminidase isozyme L is reacted with a ganglioside or a mixture of gangliosides by adding the neuraminidase to a starting solution containing the ganglioside or gangliosides and a buffer.
 5. A process to claim 4 wherein the starting solution further contains sterilized water.
 6. A process according to claim 4 wherein the amount of ganglioside or a mixture of gangliosides is not more than 50 mg per ml of the starting solution.
 7. A process according to claim 6 wherein the amount of ganglioside or a mixture of gangliosides is about 0.05 to about 2 mg per ml of the starting solution.
 8. A process according to claim 4 wherein the amount of neuraminidase isozyme L is not less than 5 mU per ml of the starting solution.
 9. A process according to claim 8 wherein the amount of neuraminidase isozyme L is about 0.1 to about 10 U per ml of the starting solution.
 10. A process according to claim 1 wherein the reaction is conducted at a temperature of about 20 to about 50° C.
 11. A process according to claim 1 wherein the reaction is conducted at a pH of about 4 to about
 8. 